Bailey, Trisha L., Hernandez-Fernaud, Juan Ramon and Gibson, Matthew I. (2021) Proline pre-conditioning of cell monolayers increases post-thaw recovery and viability by distinct mechanisms to other osmolytes. RSC Medicinal Chemistry, 12 (6). pp. 982-993. doi:10.1039/D1MD00078K ISSN 2632-8682.

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Abstract

Cell cryopreservation is an essential tool for drug toxicity/function screening and transporting cell-based therapies, and is essential in most areas of biotechnology. There is a challenge, however, associated with the cryopreservation of cells in monolayer format (attached to tissue culture substrates) which gives far lower cell yields (<20% typically) compared to suspension freezing. Here we investigate the mechanisms by which the protective osmolyte L-proline enhances cell-monolayer cryopreservation. Pre-incubating A549 cells with proline, prior to cryopreservation in monolayers, increased post-thaw cell yields two-fold, and the recovered cells grow faster compared to cells cryopreserved using DMSO alone. Further increases in yield were achieved by adding polymeric ice recrystallization inhibitors, which gave limited benefit in the absence of proline. Mechanistic studies demonstrated a biochemical, rather than biophysical (i.e. not affecting ice growth) mode of action. It was observed that incubating cells with proline (before freezing) transiently reduced the growth rate of the cells, which was not seen with other osmolytes (betaine and alanine). Removal of proline led to rapid growth recovery, suggesting that proline pre-conditions the cells for cold stress, but with no impact on downstream cell function. Whole cell proteomics did not reveal a single pathway or protein target but rather cells appeared to be primed for a stress response in multiple directions, which together prepare the cells for freezing. These results support the use of proline alongside standard conditions to improve post-thaw recovery of cell monolayers, which is currently considered impractical. It also demonstrates that a chemical biology approach to discovering small molecule biochemical modulators of cryopreservation may be possible, to be used alongside traditional (solvent) based cryoprotectants.

Item Type:	Journal Article
Subjects:	Q Science > QH Natural history Q Science > QP Physiology
Divisions:	Faculty of Science, Engineering and Medicine > Science > Chemistry Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School
Library of Congress Subject Headings (LCSH):	Cells -- Cryopreservation , Antifreeze proteins, Molecular chaperones
Journal or Publication Title:	RSC Medicinal Chemistry
Publisher:	Royal Society of Chemistry
ISSN:	2632-8682
Official Date:	1 June 2021
Dates:	Date Event 1 June 2021 Published 18 May 2021 Available 3 May 2021 Accepted
Volume:	12
Number:	6
Page Range:	pp. 982-993
DOI:	10.1039/D1MD00078K
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Open Access (Creative Commons)
Date of first compliant deposit:	21 May 2021
Date of first compliant Open Access:	24 May 2021
RIOXX Funder/Project Grant:	Project/Grant ID RIOXX Funder Name Funder ID 638661 European Research Council http://dx.doi.org/10.13039/501100000781 789182 European Research Council http://dx.doi.org/10.13039/501100000781 105627/Z/ 14/Z University of Warwick http://dx.doi.org/10.13039/501100000741 105627/Z/ 14/Z Wellcome Trust http://dx.doi.org/10.13039/100010269
Is Part Of:	1
Related URLs:	Related dataset

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